| 云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析 |
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| 引用本文： | 孙雪娃,何超,方泽民,肖亚中.云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析[J].生物工程学报,2018,34(7):1156-1168. |
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| 作者姓名： | 孙雪娃 何超 方泽民 肖亚中 |
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| 作者单位： | 安徽大学生命科学学院;现代生物制造安徽省重点实验室;安徽省微生物与生物催化工程技术研究中心 |
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| 基金项目： | 国家自然科学基金 (Nos. 31370114, 31170056) 资助。 |
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| 摘 要： | 云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。
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| 关 键 词： | 云芝，NADPH-细胞色素P450还原酶，大肠杆菌，异源表达，酶学特性 |
| 收稿时间： | 2017/12/25 0:00:00 |
Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli |
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| Xuewa Sun,Chao He,Zeming Fang and Yazhong Xiao.Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli[J].Chinese Journal of Biotechnology,2018,34(7):1156-1168. |
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| Authors: | Xuewa Sun Chao He Zeming Fang and Yazhong Xiao |
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| Institution: | 1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China; 2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China; 3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China,1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China; 2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China; 3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China,1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China; 2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China; 3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China and 1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China; 2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China; 3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China |
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| Abstract: | |
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| Keywords: | Trametes versicolor NADPH-cytochrome P450 reductase Escherichia coli heterologous expression enzymatic properties |
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